In Situ Analysis of Community Structure in Activated Sludge with 2-Hydroxy-3-Naphthoic Acid-2-Phenylanilide Phosphate and Fast Red TR In Situ Hybridization.

Abstract

Fluorescent vital staining and rRNA-targeted in situ hybridization were used to investigate the physiological activity and microbial population structures of two types of activated sludge: fresh returned sludge from a municipal wastewater treatment plant and MITI-standard sludge (a microbial source in a biodegradation test, Modified MITI Test). The ratio of esterase-active bacteria, as determined by double-staining with 4', 6-diamidino-2-phenylindole (DAPI) and 6-carboxyfluorescein diacetate (6CFDA), was 60-80% to total bacterial number in both types of activated sludge; both ratios being much higher than those of the colony forming bacteria. Microbial population structures in both types of activated sludge were determined by 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) and Fast Red TR in situ hybridization (HNPP-FISH). Approximately 50% of the DAPI-stained bacteria was detected by HNPP-FISH in wastewater treatment plant sludge and 40% in the MITI-standard sludge. The bacterial community structure in wastewater treatment plant sludge was dominated by the Flavobacterium-Cytophaga group. Characteristic coccoid cells, which fluoresced bright yellow under UV excitation after DAPI-staining, were dominant in the MITI-standard sludge.